P95--LINKING DSB REPAIR AND CELL CYCLE CHECKPOINTS

P95——连接 DSB 修复和细胞周期检查点

• 批准号: 6386489
• 负责人: John HJ Petrini
• 金额: $ 17.46万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386489
• 关键词: DNA damage; DNA repair; cell growth regulation; chromosome disorders; gene targeting; genetic models; genetically modified animals; laboratory mouse; model design /development; phenotype; radiation genetics; radiation sensitivity; syndrome

Nijmegen Breakage Syndrome (NBS) is an autosomal recessive disorder characterized by marked cancer predisposition, sensitivity to ionizing radiation, and defects in DNA damage dependent cell cycle checkpoints. Because the cellular phenotypes of NBS strongly resemble those of cells established from ataxia telangiectasia (AT) patients, this syndrome has been described as an AT variant syndrome although it is genetically distinct. We showed that NBS is attributable to deficiency in an intrinsic member of the hMre11/hRad50 protein complex, referred to herein as p95. We have established that this complex, which is uniformly distributed in the nucleus prior to irradiation, relocalizes to sites of DNA damage in the nuclei of cells treated with ionizing radiation, and is restored to uniform distribution upon completion of DNA repair. The association of the complex with DNA damage, in conjunction with the finding that cellular responses to ionizing radiation are abrogated by p95 deficiency lead to the hypothesis that the complex acts to sense and signal the presence of DNA damage in irradiated cells. In this proposal, we will examine the function of p95 to address this hypothesis, and to elucidate its potential role the repair of ionizing radiation-induced DNA damage as part of the Mre11/Rad50 complex. We propose to carry out phenotypic analyses of p95 deficient cells expressing variants of p95 to define functionally important domains of the protein. To examine phenotypes that are not assayable in cultured cells and to assess genetic interactions of p95, p95 deficient mice will be constructed. Of particular interest in this regard are the genetic and functional interactions between p95 and ATM which will be assessed by breeding of p95 and ATM deficient mice. These studies will provide important information regarding the relative contributions of p95 and ATM in the cellular and organismal responses to ionizing radiation. In this proposal, p95 is examined from three complementary perspectives. First, at the cellular level, second, at the molecular level, and third, in vivo through the derivation of a munbs1 mutant mouse.

奈梅亨断裂综合征 (NBS) 是一种常染色体隐性遗传疾病，其特征是显着的癌症倾向、对电离辐射的敏感性以及 DNA 损伤依赖性细胞周期检查点的缺陷。 由于 NBS 的细胞表型与共济失调性毛细血管扩张 (AT) 患者建立的细胞表型非常相似，因此这种综合征被描述为 AT 变异综合征，尽管它在遗传上是不同的。 我们证明NBS可归因于hMre11/hRad50蛋白复合物的内在成员（本文称为p95）的缺陷。 我们已经确定，这种复合物在辐射前均匀分布在细胞核中，重新定位到经电离辐射处理的细胞核中 DNA 损伤的位点，并在 DNA 修复完成后恢复到均匀分布。 该复合物与 DNA 损伤的关联，再加上 p95 缺陷会消除细胞对电离辐射的反应的发现，导致了这样的假设：该复合物的作用是感知并发出信号，表明受辐射细胞中存在 DNA 损伤。在本提案中，我们将检查 p95 的功能来解决这一假设，并阐明其作为 Mre11/Rad50 复合物的一部分修复电离辐射诱导的 DNA 损伤的潜在作用。 我们建议对表达 p95 变体的 p95 缺陷细胞进行表型分析，以确定该蛋白质的功能重要结构域。 为了检查在培养细胞中无法检测的表型并评估 p95 的遗传相互作用，将构建 p95 缺陷小鼠。 在这方面特别令人感兴趣的是 p95 和 ATM 之间的遗传和功能相互作用，将通过培育 p95 和 ATM 缺陷小鼠来评估。这些研究将提供有关 p95 和 ATM 在细胞和有机体对电离辐射反应中的相对贡献的重要信息。 在此提案中，从三个互补的角度对 p95 进行了审查。 首先，在细胞水平上，第二，在分子水平上，第三，通过munbs1突变小鼠的衍生在体内进行。